LABORATORY DIAGNOSIS
OF TUBERCULOSIS

CLASSIFICATION OF MYCOBACTERIA
Mycobacterium tuberculosis complex refers to a genetically
related groups of Mycobacterium species that can cause
TUBERCULOSIS [TB] in humans. It includes;
Mycobacterium tuberculosis,
Mycobacterium bovis [M.bovis, subsp bovis, M.bovis subsp.caprae and M.bovis
BCG]

SPECIMEN COLLECTION
In every clinical Microbiology sample collection
“Results are as good as Specimen” Good quality sample
is very important.
Sputum samples of good quality collected in wide mouth
sterile containers.
Quantity sufficient
Extra pulmonary samples collected in sterile containers
/syringes.

Mucoid Sample
Purulent Sample
Bloody Sample
Salivary Sample

DIAGNOSIS OF TUBERCULOSIS
Smear Microscopy
AFB Culture
Manual & Liquid Sensitivity
Molecular Diagnostic

In low income and high tuberculosis prevalence
countries, sputum smear microscopy is the only cost-
effective tool for diagnosing patients with infectious
tuberculosis and to monitor their progress in treatment.
Sputum smear microscopy is a simple, inexpensive,
appropriate technology method which is relatively easy to
perform and to read.
Classical Ziehl-Neelsen stain used-AFB
SMEAR MICROSCOPY

Smear prepared from thick purulent parts of samples.
Size of smear should be 3cmx2cm
At least 300 fields examined
Smear is positive in samples which contain 5000- 10000
bacteria /ml
Sensitivity ranges from 25%-65%
 Sensitivity increases by examination of more than one
smear
SMEAR MICROSCOPY

SIZE OF SMEAR
2 x 3
1 x 2 Uniform Smear

Good Evenness Smear
Uneven Smear
Good Thickness Smear
Too Thick Smear
Too Thin Smear

Smears reported as Positive or
Negative
Quantity of AFB observed
should be noted
Factors influencing smear
sensitivity are type of specimen,
staining technique, experience
of reader
Laboratory Quality Control
important
ZN STAINING

FLUORESCENCE MICROSCOPY
Fluorochrome stain used
Can be examined at lower magnification(40 X)
Rapid but more false positive
LED fluorescence microscopy has been evaluated- rapid
and good results, lower cost
LED attachment to microscope Primo Star iLED from Carl
Zeiss

FLUORESCENCE
MICROSCOPY

DISADVANTAGES OF SMEAR
MICROSCOPY
Needs a large no of bacilli per ml of specimen to be
detected positive
Cannot differentiate between dead and live bacilli
Cannot differentiate between Mtb and NTM
No idea of drug resistance

AFB CULTURE
GOLD STANDARD
Provides definitive diagnosis of TB
Pure growth of mycobacteria to do speciation and drug
sensitivity.
Technically demanding and complex
High level of Biosafety needed

AFB CULTURE BY L.J. MEDIA [SOLID]
Detection of 10-100 viable
bacilli/ml of specimen
Specimens have to be
decontaminated before
inoculation to remove the
normal bacterial flora.
Solid culture – Conventional LJ
method.
Mycobacteria slow growing
and hence take 2-8 weeks to
grow
LOWENSTEIN JENSEN
[L.J. ] MEDIA

LIQUID CULTURE
Many Commercial systems available- BACTEC systems
MGIT960, BacT/ALERT 3D system
Liquid culture yield significantly rapid results than solid
media and isolation rates for mycobacteria are higher
Liquid media- Middle brook 7H9 media used
MGIT system( Mycobacterial growth indicator tubes)
contains a modified Middle brook 7H9 broth with a
fluorescence quenching based oxygen sensor. Growth of
mycobacteria leads to oxygen depletion and indicator
fluoresces brightly
Cultures positive in 10-14 days

LIQUID CULTURE BY BACTEC

DRUG SENSITIVITY FOR AFB
Sensitivity to first and second line drugs available
Expensive
High degree of technical expertise and lab infrastructure
required
Rigorous quality control needed

NON COMMERCIAL METHOD
MODS [Microscopic observed drug susceptibility]
 A micro colony method in liquid culture , based on
inoculation of specimens into drug free and drug
containing media, followed by microscopic examination of
early growth .
Recommended as direct or indirect tests for rapid
screening of patients suspected of having MDR TB

CRI
( Colorimetric redox indicator)
Indirect testing methods based on the reduction of a
coloured indicator added to liquid culture medium on a
microtitre plate after exposure of M. tb strains to anti TB
drugs in vitro

NRA
(Nitrate reductase assay)
 A direct or indirect method on solid culture based on the
ability of M. tuberculosis to reduce nitrate, which is
detected by a colour reaction

SEROLOGICAL TESTS
NEGATIVE RECOMMENDATION from WHO in 2011
SHOULD NOT BE ORDERED

MOLECULAR TEST
Genotypic methods have considerable advantage of
speed, standardization of testing and reduced requirement
for Biosafety
1. LINE PROBE ASSAY
( HAINS TEST)
2. GENEXPERT

1. LINE PROBE ASSAY ( HAINS TEST)
Simultaneous identification for M.tuberculosis complex
Molecular assay for the detection of resistance to INH &
RIF of M.tuberculosis complex
By detection of most significant mutations to – inhA,
RpoB and the katG genes
Based on DNA strip technology
Can be done from positive cultures (from MGIT,
BacT/ALERT bottles or LJ)
Pulmonary samples which are smear +ve can be done
directly

Detection of multiple genes responsible for the antibiotic
resistance &
Simultaneous recognition of missing wild type gene
Also Available for Second secondline
and identification of some strain of NTM
Limitations of Genotype MTBDRplus
Needs preprocessing of samples.
Needs a PCR set up
Technically demanding
Panic of contamination
Special infrastructure required
Needs dedicated staff and space.

2. GENEXPERT [CBNAAT]
The Xpert MTB/RIF is a cartridge based nucleic acid
amplification test , automated diagnostic test that can
identify Mycobacterium tuberculosis (MTB) DNA and
resistance to Rifampicin (RIF) by Nucleic Acid
Amplification Test(NAAT).
SAMPLES;
Pulmonary samples( Sputum, BAL )
Extra pulmonary samples [Lymph node tissue and
aspirates, CSF, Pus , Gastric lavage and aspirates ( in
children) & Other Tissues]

Pulmonary samples - Xpert MTB/ Rif Sensitivity
Status Sensitivity %
Smear +ve culture +ve 98
Smear –ve culture +ve 68
People with HIV 79
People without HIV 86
Extra pulmonary samples Xpert MTB/Rif - sensitivity and specificity
Samples Sensitivity % Specificity %
Lymphnode tissue and
aspirate
84.9 92.5
CSF 79.5 98.6
Pleural fluid 43.7 98.1
Gastric lavage and
aspirations
83.8 98.1
Other tissue 81.2 98.1